1. Field of the Invention
The invention relates generally to the field of DNA methylation and more specifically to the use and detection of agents that reverse epigenetic “silencing” of genes resulting from DNA hypermethylation.
2. Background Information
DNA methylation, or the covalent addition of a methyl group to cytosine within the context of the CpG dinucleotide, has profound effects on the mammalian genome. These effects include transcriptional repression via inhibition of transcription factor binding or the recruitment of methyl-binding proteins and their associated chromatin remodeling factors, X chromosome inactivation, imprinting and the suppression of parasitic DNA sequences. DNA methylation is also essential for proper embryonic development; however, its presence can add an additional burden to the genome. Normal methylation patterns are frequently disrupted in tumor cells with global hypomethylation accompanying region-specific hypermethylation. When these hypermethylation events occur within the promoter of a tumor suppressor gene they may silence the gene and provide the cell with a growth advantage in a manner akin to deletions or mutations. Furthermore, DNA methylation may be an important player in both DNA repair and genome stability.
DNA methylation at the 5-position of cytosine in CpG dinucleotides is an important aspect of physiological processes, such as embryonic development, X chromosome inactivation, imprinting, and transcriptional regulation. While CpG dinucleotides are generally methylated throughout the genome of normal somatic cells, CpG islands (CGIs), clusters of CpG dinucleotides in gene regulatory regions, are usually unmethylated.
Epigenetic gene “silencing” occurs in cancer cells. Of all the somatic genome changes that accumulate during the pathogenesis of human cancer, only changes in DNA methylation appear to occur consistently (virtually all cases), to arise early (first appearing in preneoplastic lesions), and to be potentially reversible (the DNA sequence remains intact). One such change in DNA methylation, increased CpG dinucleotide methylation at CpG islands encompassing the transcriptional regulatory regions of many genes, leads to the transcriptional “silencing” of critical cancer genes. Aberrant hypermethylation of CGIs and subsequent transcriptional repression is one of the earliest and most common somatic genome alterations in multiple human cancers.
CpG island hypermethylation has been reported to inhibit gene transcription by interfering with the binding and/or function of transcriptional transactivators, or by recruiting 5−m CpG-binding domain (MBD) family proteins capable of mediating transcriptional repression via effects on chromatin structure. As an example, for the GSTP1 CpG island hypermethylated in cancers, such as prostate, breast, and liver cancers, the MBD family protein MBD2 has been found responsible for methylation-associated “silencing” of gene transcription.
Somatic CpG island hypermethylation and associated gene “silencing” may be effectively targeted for rational cancer treatment and prevention. One strategy, under current clinical development, features the use of inhibitors of DNA methyltransferases (DNMTs), such as 5-aza-cytdine, 5-aza-deoxycytidine, zebularine, procainamide, or hydralazine, to reduce 5−mCpG density at the CpG island sequences in dividing cancer cells. Another approach, also under active clinical development, has been the use of inhibitors of histone deacetylases (HDACs), such as sodium phenylbutyrate, valproic acid, or suberoylanilide hydroxamic acid (SAHA), to limit the formation of repressive chromatin conformation near the genes caring abnormally methylated CpG islands.
Nucleoside analog inhibitors of DNMTs, such as 5-az-cytidine (5-aza-C) and 5-aza-deoxycytidine (5-aza-dC), have been widely used in attempts to reverse abnormal DNA methylation changes in cancer cells and restore “silenced” gene expression. Unfortunately, despite some apparent successes using pre-clinical models and some promising results in early clinical trials (Table 1), the clinical utility of these compounds for cancer has not yet been fully realized and the drugs have not yet been approved by the U.S. Food and Drug Administration (F.D.A.) for any indication.
TABLE 1Nucleoside DNMT Inhibitors and Solid Tumors% Res-TumorAgentnDose/Schedule/Routeponsebreast ca5-aza-C11300-700 mg/M2 over 8 days63iv5-aza-C31600 mg/M2 over 10 days iv65-aza-C4275-850 mg/M2 over2510 days sq5-aza-C1.6 mg/kg over 10 days iv17ovarian ca5-aza-C4275-850 mg/M2 over2510 days sq5-aza-dC24225 mg/M2 over 10 days iv8colon ca5-aza-C2745-aza-C6300-700 mg/M2 over3310 days iv5-aza-C4275-850 mg/M2 over 8 days0sq5-aza-dC42225 mg/M2 over 1 day iv0lung ca5-aza-dC15200-600 mg/M2 over 1 day20ivprostate ca5-aza-dC14225 mg/M2 over 1 day iv16melanoma5-aza-C5300-700 mg/M2 over 8 days40iv5-aza-dC18225 mg/M2 over 1 day iv0dh-5-aza-C405 g/M2 over 1 day cl20mesotheliomadh-5-aza-C411500 mg/M2 over 5 days cl17dh-5-aza-C291500 mg/M2 over 5 days cl17
One of the limitations of the nucleoside analog DNMT inhibitors in clinical trials has been treatment-associated side effects, such as myelotoxicity with resultant neutropenia and thrombocytopenia, which are characteristic of other nucleoside analogs in general, including nucleoside analogs that are not DNMT inhibitors.
Another concern about the use of nucleoside analogs as DNMT inhibitors has been that incorporation of the nucleoside analogs into genomic DNA might lead to mutations and/or cancer development. Procainamide, a drug approved by the F.D.A. for the treatment of cardiac arrhythmias, and hydralazine, a drug approved for the treatment of hypertension, are non-nucleoside analogs that both also appear to inhibit DNMTs. However, long-term use of either of these drugs caries a risk of drug-induced lupus, more commonly in women than in men. In animal models, both 5-aza-C and procainamide appear to trigger autoimmunity, though whether or not autoimmunity is an unavoidable side effect of DNMT inhibition is not known. Finally, mice carrying one disrupted DNMT1 allele and one hypomorphic DNMT1 allele, resulting in 10% of normal DNMT activity, have been reported to exhibit genomic instability and to develop T-cell lymphomas, hinting that therapeutic reductions in 5−mCpG dinucleotides might promote the appearance of certain cancers (eg. lymphomas) while attenuating the appearance of others. Thus, the clinical use of DNMT inhibitors is likely to be limited by both mechanism-based and mechanism-independent side effects.
Like DNMT inhibitors, HDAC inhibitors have also exhibited promising preclinical activity in cancer models. HDAC inhibitors under clinical development include sodium phenyl butyrate (and other butyrates), valoproic acid, suberoylanilide hydroxamic acid (SAHA), pyroxamide, N-acetyl dinaline (CI-994), and depsipeptide. However, the early clinical experience with these agents suggests that side effects, such as nausea, vomiting, diarrhea, fatigue, edema, etc., can occur, though severe adverse events appear rare. In addition to DNMT inhibitors and HDAC inhibitors given as single agents, combinations of DNMT inhibitors and HDAC inhibitors also appear to have intriguing activity in preclinical models. Whether combinations of the currently available collection of DNMT inhibitors and HDAC inhibitors can reactivate silenced cancer genes, without unacceptable toxicity, in human clinical trials, has not yet been determined.
In addition to treatment and prevention of cancer, reactivation of silenced genes may be useful for treatment of other diseases, such as sickle cell anemia. Sickle cell anemia is caused by a point mutation in the beta-globin gene (HBb). Dimers of this mutant form of HBb multimerize with dimers of alpha-globin (Hba) to make sickle hemoglobin (HBs). HBs is prone to polymerization, causing sickling of red blood cells, and subsequent aberrant interactions between the sickled red blood cells, immune cells, and endothelial cells that result in a complex spectrum of disease manifestations. Reactivation of the gamma-globin gene, presents a useful strategy in treatment of sickle cell disease.